DESCRIPTION: With the ability to differentiate into multiple mesenchymal lineages, human mesenchymal stem cells (hMSCs) are important regulators of tissue regeneration and have also been extensively explored as cell-based clinical therapies. However, diseased microenvironments can direct hMSCs into maladaptive lineages and controlling cell fate is still a major challenge for many of these therapies. While past studies have revealed the important roles of chemical cues in directing hMSC differentiation, the effects of physical cues from the extracellular microenvironment remain less understood. This is partly due to traditional culture of hMSCs on supra- physiologically stiff plastic plates, but also the need for better tools to de-convolute the complexities of matrix signaling. This application aims to exploit poly(ethylene glycol)-based hydrogels with phototunable elasticities, and use these bioscaffolds as hMSC cell culture substrates to study quantitatively how mechanical cues may regulate cell fate in a time- and dose-dependent manner. We hypothesize that high mechanical doses from rigid culture substrates will persistently activate the mechanosensors YAP/TAZ and bias differentiation and reduce hMSC multipotency. To better understand how extracellular physical cues may lead to irreversible changes through a YAP/TAZ-mediated mechanical dosing process, ChIP-seq experiments will be performed against YAP to locate specific genes that are associated with different dosing conditions. Collectively, these studies should help to improve the design of biomaterial delivery vehicles for hMSC therapies, and provide insight into major intracellular signaling machinery that may be involved in translating and quantifying extracellular mechanical dosing into genomic information that may direct stem cell fate.